Isolation of myenteric and submucosal plexus from mouse gastrointestinal tract and subsequent flow cytometry and immunofluorescence

Abstract

Summary The myenteric plexus is located between the longitudinal and circular layers of muscularis externa in the gastrointestinal tract. It contains a large network of enteric neurons that form the enteric nervous system (ENS) and control intestinal functions, such as motility and nutrient sensing. This protocol describes the method for physical separation (peeling) of muscularis and submucosal layers of the mouse intestine. Subsequently, the intestinal layers are then processed for flow cytometry and/or immunofluorescence analysis. For complete details on the use and execution of this profile, please refer to Ahrends et al. (2021) .

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system

“…The muscularis externa containing myenteric plexuses was prepared from mouse GITs as previously described with some modification ( 65 , 66 ). For HILIC-MS/MS analysis, mouse GIT segments were cut open along the attachment line of the mesentery and then placed onto a cold surface with the muscularis externa facing up.…”

“…Immunostaining of duodenal muscularis externa was performed as previously described with minor modifications ( 65 , 66 ). Briefly, duodenal muscularis externa isolated from WT and IP6K2 −/− mice were blocked with 3% bovine serum albumin blocking solution containing the corresponding isotype control antibodies (IgG2a, R&D systems, Cat# MAB003; IgG2b, Dako) for 2 days after fixation with 4% paraformaldehyde overnight.…”

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in enteric nervous system

“…Immunostaining of duodenal muscularis externa was performed as previously described with minor modifications (Fujita et al, 2018; Ahrends et al, 2022). Briefly, duodenal muscularis externa isolated from WT and IP6K2 -/- mice were blocked with 3% BSA blocking solution containing the corresponding isotype control antibodies for 2 days after fixation with 4% PFA overnight.…”

“…The muscularis externa containing myenteric plexuses was prepared from mouse GITs as previously described with some modification (Fujita et al, 2018; Ahrends et al, 2022). For HILIC-MS/MS, mouse GIT segments were cut open along the attachment line of the mesentery and then placed onto a cold surface with the muscularis externa facing up.…”

“…al, 2018;Ahrends et al, 2022). For HILIC-MS/MS, mouse GIT segments were cut open along the attachment line of the mesentery and then placed onto a cold surface with the muscularis externa facing up.…”